Project description:This project examines the impact of different growth media on the protein compositions of OMV and membrane of Bacteroides thetaiotaomicron.
Project description:This project examines the impact of different growth media on the protein compositions of membranes of Bacteroides thetaiotaomicron.
Project description:The trypanosomatid protozoan parasite Leishmania has a significant impact on human health globally. Understanding the pathways associated with virulence within this significant pathogen is critical for identifying novel vaccination and chemotherapy targets. Within this study we leverage an ultradeep proteomic approach to improve our understanding of two virulence associated genes in Leishmania; the Golgi Mannose/Arabinopyranose/Fucose nucleotide-sugar transporter LPG2, and the mitochondrial fucosyltransferase FUT1. Using deep peptide fractionation followed by complementary fragmentation approaches with higher energy collisional dissociation (HCD) and Electron-transfer dissociation (ETD) allowed the identification of over 6500 proteins, nearly doubling the experimentally observed Leishmania major proteome. This deep proteomic analysis revealed significant quantitative differences in both lpg2- and fut1s mutants with FUT1-dependent changes linked to marked alterations within mitochondrial associated proteins while LPG2-dependent changes impacted multiple aspects of the secretory pathway. While FUT1 has been shown to fucosylate peptides in vitro, no evidence for protein fucosylation was identified within our ultradeep analysis nor did we observe fucosylated glycans within Leishmania glycopeptides isolated using HILIC enrichment. Combined this work provides a critical proteomic resource for the community on the observable Leishmania proteome as well as highlights phenotypic changes associated with LPG2/FUT1 which may guide the development of future therapeutics.
Project description:Comparative proteomics of Bacteroides thetaiotaomicron samples comparing the total membrane (TM) and outer membrane vesicles (OMV) of WT B. thetaiotaomicron and delta 4364
Project description:This work explore the optimisation of PglS to allow the improved production of glycoproteins modified with Streptococcus agalactiae associated capsule glycans.
Project description:Protein glycosylation is increasingly recognized as a common protein modification across bacterial species. Within members of the Neisseria genus O-linked protein glycosylation plays important roles in virulence and antigenic variation yet our understanding of the substrates of glycosylation are limited. Recently it was identified that even closely related Neisserial species can possess O-oligosaccharyltransferases, pglOs, that possess varying glycosylation specificities suggesting that distinct targeting activities may impact both the glycoprotome as well as the proteome of Neisserial species. Within this work we explore this concept using of Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) fractionation and Data-Independent Acquisition (DIA) to allow the characterization of differences in the glycoproteomes and proteomes within N. gonorrhoeae strains expressing differing pglO alleles. We demonstrate the utility of FAIMS to expand the known glycoproteome of N. gonorrhoeae and enable comparative glycoproteomics of a recently reported panel of N. gonorrhoeae strains expressing different pglO allelic chimeras (15 pglO enzymes) with unique substrate targeting activities. Combining glycoproteomic insights with DIA proteomics we demonstrate that alterations within pglO alleles have widespread impacts on the proteome of N. gonorrhoeae yet lead to minimal effects on the abundance of glycoproteins. Additionally, while DIA analysis can allow occupancy to be inferred by the absence or presence of peptides known to be modified, we observe a poor correlation between DIA measurements of non-modified versions of glycopeptides and glycoproteomic analysis. Combined this work expands our understanding of the N. gonorrhoeae glycoproteome and supports that the expression of different pglO alleles appears to drive proteomic changes independent of the glycoproteins targeted for glycosylation.
Project description:Protein glycosylation is increasingly recognized as a common protein modification across bacterial species. Within members of the Neisseria genus O-linked protein glycosylation plays important roles in virulence and antigenic variation yet our understanding of the substrates of glycosylation are limited. Recently it was identified that even closely related Neisserial species can possess O-oligosaccharyltransferases, pglOs, that possess varying glycosylation specificities suggesting that distinct targeting activities may impact both the glycoprotome as well as the proteome of Neisserial species. Within this work we explore this concept using of Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) fractionation and Data-Independent Acquisition (DIA) to allow the characterization of differences in the glycoproteomes and proteomes within N. gonorrhoeae strains expressing differing pglO alleles. We demonstrate the utility of FAIMS to expand the known glycoproteome of N. gonorrhoeae and enable comparative glycoproteomics of a recently reported panel of N. gonorrhoeae strains expressing different pglO allelic chimeras (15 pglO enzymes) with unique substrate targeting activities. Combining glycoproteomic insights with DIA proteomics we demonstrate that alterations within pglO alleles have widespread impacts on the proteome of N. gonorrhoeae yet lead to minimal effects on the abundance of glycoproteins. Additionally, while DIA analysis can allow occupancy to be inferred by the absence or presence of peptides known to be modified, we observe a poor correlation between DIA measurements of non-modified versions of glycopeptides and glycoproteomic analysis. Combined this work expands our understanding of the N. gonorrhoeae glycoproteome and supports that the expression of different pglO alleles appears to drive proteomic changes independent of the glycoproteins targeted for glycosylation.